Hormonal control of adenylate cyclase is mediated by GTP-binding proteins, stimulation via Gs, inhibition via Gi. Cholera toxin and pertussis toxin disrupt hormonal control of adenylate cyclase by ADP-ribosylating Gs and Gi, respectively. GTP-binding proteins appear to be involved in other transmembrane signalling events as well. One of these, transducin, and reginal GTP-binding protein that couples the light receptor rhodopsin to a cGMP phosphodiesterase, serves as a substrate for cholera toxin and pertussis toxin. Because transducin shares functional and structural homology with other G proteins it is useful for the study of the toxins' mechanisms. In these studies, we have determined the site of pertussis toxin catalyzed ADP-ribosylation of transducin. Pertussis toxin catalyzed the incorporation of radiolabel from [U-14C-adenine]-NAD into the Alpha subunit of holotransducin from bovine retinas. Maximal incorporation was 0.3 mol ADP-ribose/mol transducin. A tryptic peptide containing ADP-ribose was purified by sequential chromatography on TSK-DEAE, phenylboronate polyacrylamide and Ultrapore C3. The amino acid content was analyzed and the peptide sequence determined to be glu-asn-leu-lys-asp-x-gly-leu-phe, in agreement with the amino acid analysis which included, in addition, cysteine. This corresponds to the recently published transducin cDNA sequence glu-346 through phe-354 which shows residue 351 to be cysteine. HPLC of the acetic acid eluate of the sequencing resin showed two radioactive species, one eluting between NAD and ADP-ribose, presumably ADP-ribosyl cysteine, and the other coincident with 5'-AMP. Amino acid analysis of the eluate disclosed the presence of cysteine. We conclude that cys-351, not an asparagine as previously reported is the site of pertussis toxin-catalyzed ADP-ribosylation of transducin. Because of transducin's sequence homology with Gs and Gi, this is most likely the modification site in these proteins as well. This cysteine is apparently critical for the coupling of inhibitory receptors to adenylate cyclase.